The long-term objective of this grant is to investigate the mechanisms that regulate the productive life cycle of human papillomaviruses (HPV) with a focus on the action of the E5, E1^E4 and E6 proteins. HPVs infect basal epithelial cells and stably maintain episomes. As these cells divide and undergo differentiation, the productive replication of viral genomes as well as activation of late gene expression is induced. In order to understand the pathways regulating the viral life cycle, a genetic analysis was performed in the context of complete viral genomes. The E5 and E1^E4 proteins were found to be necessary for full activation of differentiation-dependent late viral functions. In addition, a series of potential binding partners of E5 were identified. In additional studies we determined that the E6 proteins from both high and low risk HPV types are needed for the stable maintenance of episomes in undifferentiated cells. The binding of E6TP1 and PDZ- domain containing proteins were both shown to be important for this ability. Further studies demonstrated that HPV gene products activated caspases upon differentiation and this was necessary for genome amplification. The E6 protein activated anti-apoptotic proteins that likely act to block premature apoptosis in differentiating cells. This renewal application centers on further investigation of the roles of E5, E1^E4 and E6 in the differentiation dependent life cycle. I propose to address the following questions in this renewal application. 1) How do the high-risk E5 proteins activate the differentiation-dependent late phase of the HPV life cycle? 2) What functions do the E1^E4 proteins provide in the late phase of the HPV life cycle? What are their targets of action? Do these proteins function synergistically with E5? 3) How does E6 facilitate maintenance of episomes in undifferentiated cells? What activities does E6 control in differentiated cells?